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Biology

야마나카 인자의 과발현에 의해 능성에 성인 체세포 세포의 생체 내 재 프로그램

Published: December 17, 2013 doi: 10.3791/50837
Automatic Translation
1, 1, 1, 2
1Nanomedicine Lab, UCL School of Pharmacy, University College London, 2Nanomedicine Lab, Faculty of Medical & Human Sciences, University of Manchester

Abstract

정의 요인 강제 식으로 다 능성 상태로 체세포의 재 프로그램의 결과 유도 만능 줄기 (IPS) 세포는 재생 의학을위한 새로운 기회를 제공하고 있습니다. IPS 세포의 이러한 임상 적용은 주로 기존 프로그래밍 방법론의 열악한 효율성 및 주입시 종양을 형성하기 위해 생성 된 만능 줄기 세포의 위험에, 지금까지 제한되어있다.

우리는 다 능성으로 체세포의 프로그래밍은 프로그래밍 인자의 유전자 전달에 의해 생체 내에서 달성 될 수 있다는 가설을 세웠다. 효율적으로 생체 내에서 세포를 재 프로그래밍하기 위해, 야마나카 (OKSM) 전사 인자의 높은 수준은 표적 조직에서 발현 될 필요가있다. 이것은 표적 조직에 따라 다른 바이러스 성 또는 비 바이러스 성 유전자 벡터를 이용함으로써 달성 될 수있다. 특정 연구에서, 플라스미드 DNA의 유체 역학적 꼬리 정맥 (HTV) 주사를 제공하는 데 사용되었다마우스 간세포에 OKSM 요인. 이 증거의 증거 성인의 생체 프로그래밍, 높은 효율과 빠른 반응 속도와 능성 상태를 향해 체세포를 제공했다. 한층 더 종양이나 기형 종 형성은 동일 반응계에서 관찰되지 않았다.

이는 생체 내에서 체세포를 재 프로그램하는 프로토콜을 수행하고 향후 다른 조직 유형에 적용될 수있는 시험관에 비해 효율적으로 안전하게, 빠르게 향상된 능성을 유도하는 잠재력 접근법을 제안하고, 수 있다는 결론을 내릴 수있다.

Materials

Name Company Catalog Number Comments
pCX-OKS-2A pDNA Addgene 19771 Obtained from Addgene as bacterial stab, plasmid production performed in Plasmid Factory (Germany)
pCX-cMyc Addgene 19772 Obtained from Addgene as bacterial stab, plasmid production performed in Plasmid Factory (Germany)
0.22 μm filter Milliopore SLGP033RS
Isoflurane Abbott B505
HBSS buffer Sigma-Aldrich H6648 Ca2+ and Mg2+ free, with bicarbonate
Liver Digest Medium Gibco 17703-034
Hepatocyte Wash Medium Gibco 17704-024
100 μm cell strainer BD Biosciences 352360
Nucleospin RNA II kit Macherey-Nagel 740955.25 Kit for RNA isolation from cells and tissues
iScript cDNA synthesis kit Bio-Rad 170-8890
iO SYBR Green Supermix Bio-Rad 170-8880
Oct3/4 forward primer Sigma sequence given in comments TGAGAACCTTCAGGAGATATGCAA
Oct3/4 reverse primer Sigma sequence given in comments CTCAATGCTAGTTCGCTTTCTCTTC
Sox2 forward primer Sigma sequence given in comments GGTTACCTCTTCCTCCCACTCCAG
Sox2 reverse primer Sigma sequence given in comments TCACATGTGCGACAGGGGCAG
C-myc forward primer Sigma sequence given in comments CAGAGGAGGAACGAGCTGAAGCGC
C-myc reverse primer Sigma sequence given in comments TTATGCACCAGAGTTTCGAAGCTGTTCG
Nanog forward primer Sigma sequence given in comments CAGAAAAACCAGTGGTTGAAGACTAG
Nanog reverse primer Sigma sequence given in comments GCAATGGATGCTGGGATACTC
Ecat1 forward primer Sigma sequence given in comments TGTGGGGCCCTGAAAGGCGAGCTGAGAT
Ecat1 reverse primer Sigma sequence given in comments ATGGGCCGCCATACGACGACGCTCAACT
Rex1 forward primer Sigma sequence given in comments ACGAGTGGCAGTTTCTTCTTGGGA
Rex1 reverse primer Sigma sequence given in comments TATGACTCACTTCCAGGGGGCACT
Alb forward primer Sigma sequence given in comments GTTCGCTACACCCAGAAAGC
Alb reverse primer Sigma sequence given in comments CCACACAAGGCAGTCTCTGA
Aat forward primer Sigma sequence given in comments CAGAGGAGGCCAAGAAAGTG
Aat reverse primer Sigma sequence given in comments ATGGACAGTCTGGGGAAGTG
Trf forward primer Sigma sequence given in comments ACCATGTTGTGGTCTCACGA
Trf reverse primer Sigma sequence given in comments ACAGAAGGTCCTTGGTGGTG
B actin forward primer Sigma sequence given in comments GACCTCTATGCCAACACAGT
B actin reverse primer Sigma sequence given in comments AGTACTTGCGCTCAGGAGGA
BD Cytofix fixation buffer BD Biosciences 560585
1x BD Perm/Wash buffer BD Biosciences 560585
anti-mouse OCT4-PerCP-Cy5.5 BD Biosciences 560585
anti-mouse Nanog-PE BD Biosciences 560585
2-Methylbutane Sigma-Aldrich M32631
X100-Triton Sigma-Aldrich X100-500ML
Goat serum Sigma-Aldrich G9023
BSA Sigma A2153
rabbit pAb anti-OCT4 Abcam ab19857 Use at a concentration of 3 μg/ml
rabbit pAb anti-SOX2 Abcam ab97959 Use at a concentration of 1 μg/ml
rabbit pAb anti-Nanog Abcam ab80892 Use at a concentration of 1 μg/ml
mouse mAb anti-SSEA1 Abcam ab16285 Use at a concentration of 20 μg/ml
goat pAb anti-rabbit IgG labeled with Cy3 Jackson ImmunoResearch Laboratories Inc. 111-165-003-JIR Use at a concentration of 1/250
goat pAb anti-mouse IgG labeled with Cy3 Jackson ImmunoResearch Laboratories Inc. 115-165-003-JIR Use at a concentration of 1/250
VECTASHIELD mounting medium with DAPI Vector Laboratories H-1200
BCIP/NBT liquid substrate system Sigma B1911
Aqueous mounting medium Sigma
16% Paraformaldehyde Fisher AA433689M Stock solution is 16%, use it at 4%
Hematoxylin and eosin Sigma GH53/HT110216
PAS staining Sigma 395B

DOWNLOAD MATERIALS LIST

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야마나카 인자의 과발현에 의해 능성에 성인 체세포 세포의 <em>생체 내 재</em> 프로그램
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Cite this Article

Yilmazer, A., de Lázaro, I.,… More

Yilmazer, A., de Lázaro, I., Bussy, C., Kostarelos, K. In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors. J. Vis. Exp. (82), e50837, doi:10.3791/50837 (2013).

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